Postmortem examination and toxicological analysis for acute metonitazene intoxication in Japan: A case report.

Benzoimidazole analgesics (Nitazenes, NZs) are opioid receptor agonists that exhibit very strong pharmacological effects at minute doses, and their abuse has recently become a concern worldwide. Although no deaths involving NZs had been reported in Japan to date, we recently experienced an autopsy case of a middle-aged man who was determined to have died from poisoning by metonitazene (MNZ), a type of NZs. There were traces of suspected illegal drug use around the body. Autopsy findings were consistent with acute drug intoxication as the cause of death, but it was difficult to identify the causative drugs by simple qualitative drug screening. Analysis of compounds recovered from the scene where the body was found identified MNZ, and its abuse was suspected. Quantitative toxicological analysis of urine and blood was performed using a liquid chromatography high-resolution tandem mass spectrometer (LC-HR-MS/MS). Results showed that MNZ concentrations in blood and urine were 6.0 and 5.2 ng/mL, respectively. Other drugs detected in blood were within therapeutic ranges. Quantitated blood MNZ concentration in the present case was in the similar range as those reported in overseas NZs-related deaths. There were no other findings that could have contributed to the cause of death, and the decedent was judged to have died of acute MNZ intoxication. Emergence of NZs distribution has been recognized in Japan similarly to overseas; early investigation of their pharmacological effects as well as crackdown on their distribution is strongly desired.

Human and rat microsomal metabolites of N-tert-butoxycarbonylmethamphetamine and its urinary metabolites in rat.

PURPOSE: N-tert-Butoxycarbonylmethamphetamine (BocMA), a masked derivative of methamphetamine (MA), converts into MA under acidic condition and potentially acts as a precursor to MA following ingestion. To investigate the metabolism and excretion of BocMA, metabolism tests were conducted using human liver microsomes (HLM), rat liver microsomes (RLM) and rat. METHODS: BocMA metabolites were analyzed after 1000-ng/mL BocMA incubation with microsomes for 3, 8, 13, 20, 30, and 60 min. Rats were administered intraperitoneal injections (20 mg/kg) of BocMA and their urine was collected in intervals for 72 h. Metabolites were detected by liquid chromatography-tandem mass spectrometry with five authentic standards. RESULTS: Several metabolites including 4-hydroxy-BocMA, N-tert-butoxycarbonylephedrine and N-tert-butoxycarbonyl-cathinone were detected for HLM and RLM. In the administration test, three glucuronides of hydroxylated metabolites were detected. The total recovery values of BocMA and the metabolites during the first 72 h accounted for only 0.3% of the administered dose. Throughout the microsomal and administration experiments, MAs were not detected. CONCLUSION: Hydroxylation, carbonylation and N-demethylation were proposed as metabolic pathways. However, BocMA and phase I metabolites were hardly detected in urine. This study provides useful information to interpret the possibility of BocMA intake as the cause of MA detection in biological sample.

Incorporation of Methoxyphenamine into Hair in Early Stage after Intake.

In order to investigate the incorporation behavior of drugs into hair in early stage (within 24 h) after intake, time-course changes in drug distribution in black hair were carefully analyzed after a single oral administration of methoxyphenamine (MOP), a non-regulated analog of methamphetamine. Single-hair specimens collected by plucking with the roots intact at appropriate intervals post-intake were each divided into 1-mm segments from the proximal end, and MOP in each segment was determined by a validated liquid chromatography-tandem mass spectrometry procedure. At 10 min after intake, MOP was not detected in any of the segments. MOP became detectable 30 min after intake in the hair bulb (0-1-mm segment from the proximal end) and 1 h after intake in the upper dermis zone (1-2-mm to 4-5-mm segments). The amount of MOP in the hair bulb increased rapidly over 3 h after intake and reached a maximum concentration of ∼100-900 pg/1-mm single hair (11-95 ng/mg) around 3-10 h after intake, whereas that in the upper dermis zone increased at a more gradual pace over 24 h and reached a plateau at ∼30-100 pg/1-mm hair (3-11 ng/mg). These differences can be attributed to the different incorporation mechanisms of the drug. Results from this study can further elucidate the drug incorporation mechanism, which is crucial for accurately interpreting results in hair analyses. Our findings also suggest that hair drug analysis with special attention to the hair root can serve as a useful complementary approach to urine- and blood-based testing in the field of forensic toxicology.

Dehydration-fragmentation mechanism of cathinones and their metabolites in ESI-CID.

Various cathinone-derived designer drugs (CATs) have recently appeared on the drug market. This study examined the mechanism for the generation of dehydrated ions for CATs during electrospray ionization collision-induced dissociation (ESI-CID). The generation mechanism of dehydrated ions is dependent on the amine classification in the cathinone skeleton, which is used in the identification of CATs. The two hydrogen atoms eliminated during the dehydration of cathinone (primary amine) and methcathinone (secondary amine) were determined, and the reaction mechanism was elucidated through the deuterium labeling experiments. The hydrogen atom bonded to the amine nitrogen was eliminated with the proton added during ESI, in both of the tested compounds. This provided evidence that CATs with tertiary amine structures (such as dimethylcathinone and α-pyrrolidinophenones [α-PPs]) do not undergo dehydration. However, it was shown that the two major tertiary amine metabolites (1-OH and 2″-oxo) of CATs generate dehydrated ions in ESI-CID. The dehydration mechanisms of the metabolites of α-pyrrolidinobutiophenone (α-PBP) belongs to α-PPs were also investigated. Stable-isotope labeling showed the dehydration of the 1-OH metabolite following a simple mechanism where the hydroxy group was eliminated together with the proton added during ESI. In contrast, the dehydration mechanism of the 2″-oxo metabolite involved hydrogen atoms in three or more locations along with the carbonyl group oxygen, indicating that dehydration occurred via multiple mechanisms likely including the rearrangement reaction of hydrogen atoms. These findings presented herein indicate that the dehydrated ions in ESI-CID can be used for the structural identification of CATs.

Effects of lipophilicity and functional groups of synthetic cannabinoids on their blood concentrations and urinary excretion.

The influence of lipophilicity and functional groups of synthetic cannabinoids (SCs) on their blood concentrations and urinary excretion has been studied by analyzing blood and urine specimens sampled from drivers who were involved in a car crashes under the influence of SCs. A total of 58 specimens (26 urine and 31 blood specimens), sampled within 13h of the occurrence, were analyzed by liquid chromatography-tandem mass spectrometry. Fifteen SCs were detected in those specimens; the SCs detected were categorized as follows: Class 1, Naphthoyl/Benzoyl indole (EAM2201 and three other analogs); Class 2, Indole-3-carboxylate/carboxamide containing naphthol/quinol (5F-PB-22 and four other analogs); and Class 3, Indazole-3-carboxamide containing valine/tert-leucine derivative (5F-AMB and five other analogs). The calculated lipophilicity index log P, the octanol/water participation coefficient, of those SCs in Classes 1, 2, and 3 ranged between 5.01-8.14, 5.80-6.74 and 2.29-3.81, respectively. Class 3 SCs were detectable in 12 out of 13 urine specimens, but those in Classes 1 and 2 were not detected in urine. Our analytical results indicated that the boundary line for their detectability in urine lies between log P 4 and 5. The blood concentrations of Class 3 SCs varied widely (0.0036-31ng/ml) depending on their log P, while much smaller variation was observed among those in Class 2 (0.10-5.0ng/ml).

Incorporation of zolpidem and methoxyphenamine into white hair strands after single administrations: Influence of hair pigmentation on drug incorporation.

In order to investigate the influence of pigmentation on the incorporation of drugs into hair, time-course changes in drug distribution along non-pigmented (white) hairs as well as pigmented (black) hairs plucked from the same subject was observed following single administrations of two basic drugs with different properties, zolpidem and methoxyphenamine. These drugs in 1-mm sections of single hair specimens were each determined by a liquid chromatography-tandem mass spectrometric procedure. During the early stage (12-36 h) after intake, for black hairs, both drugs were detected over the entire area of hair root (4-5 mm in length), in which notable concentration of these drugs in the hair bulb (0-1-mm segment from the bottom of hair root, Region 1) and lower concentrations in the upper dermis zone (1-2-mm to 3-4-mm or to 4-5-mm segments, Region 2) were commonly observed. Meanwhile, for white hairs, high drug concentrations in Region 1 as detected in black hairs were not observed although only small amounts of these drugs were detected over Region 2. Subsequent time-course changes in the concentration of drugs in hair demonstrated that the drugs once incorporated into white hair via Region 2 decreased gradually over the period from 24 h to 35 days after intake, but those of black hairs remained almost unchanged. These findings revealed here suggest that hair pigments have two important roles in the distribution of drugs: (1) incorporation of drugs into hair via Region 1, and (2) retention of already incorporated drugs in the hair tissue. These findings would be useful for discussing individual drug-use history based on hair analysis in the forensic fields.

Metabolism of α-PHP and α-PHPP in humans and the effects of alkyl chain lengths on the metabolism of α-pyrrolidinophenone-type designer drugs.

PURPOSE: This study aims to investigate the urinary metabolites of two common α-pyrrolidinophenones (PPs), α-pyrrolidinohexiophenone (α-PHP) and α-pyrrolidinoheptanophenone (α-PHPP). This report also aims to discuss the effects of alkyl chain lengths on the metabolism of PPs. METHODS: Urinary metabolites of α-PHP and α-PHPP have been investigated by analyzing urine samples from their users (n = 13 each) by liquid chromatography-high-resolution tandem mass spectrometry using reference standards of the metabolites synthesized in our laboratory. RESULTS AND CONCLUSIONS: For both drugs, metabolites via reduction of the keto moiety (1-OH metabolites) and via oxidation of the pyrrolidine ring (2″-oxo metabolites) were identified, and those via oxidation of the terminal (ω) or penultimate (ω-1) positions of the alkyl chain were tentatively identified. Quantitative analysis indicated oxidation of the pyrrolidine ring to be the major metabolic pathway for α-PHP (side chain R: hexyl), but ω or ω-1 oxidation was the major metabolic pathway for α-PHPP (R: heptyl). Comparison of their metabolic profiles with those of analogs with a longer or shorter side chain (studied previously for R: butyl, pentyl, and octyl) revealed that the alkyl chain length strongly influences the metabolic pathway. In addition, to the best of our knowledge, this is the first report describing the quantification of metabolites of α-PHP and α-PHPP in authentic urine specimens collected from the users using their reference standards synthesized.

Incorporation of Zolpidem into Hair and Its Distribution after a Single Administration.

To obtain fundamental information on the drug incorporation into hair, time-course changes in drug distribution along single-strand hair were observed after a single oral administration of zolpidem (ZP), one of the most frequently used hypnotic agents. Quantitative sectional hair analyses of 1-mm segments were performed for each single-strand hair using a validated LC-MS/MS procedure. ZP was detected in all specimens plucked at 10 and 24 hours after a single dose, and the distribution ranged over the whole hair root (4-5 mm in length). A significantly high concentration of ZP was detected in the hair bulb region, whereas much lower concentrations were widely observed in the upper part of the hair root of those samples; this suggested that the incorporation of ZP occurred in two regions, mainly in the hair bulb and to a lesser extent in the upper dermis zone. The ZP-positive area formed lengths of up to 10-12 mm after a single administration, indicating that its incorporation from the hair bulb would continue for about 2 weeks. Time-course changes in the ZP concentration in the hair root additionally revealed that only a small portion of ZP that initially concentrated in the bulb was successively incorporated into the hair matrix and moved toward the keratinized region as hair grew. These findings should be taken into account upon discussing individual drug-use history based on hair analysis. The matrix-assisted laser desorption/ionization mass spectrometry imaging of ZP in the same kinds of hair specimens was also successfully achieved.

Enantioseparation of the carboxamide-type synthetic cannabinoids N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamide and methyl [1-(5-fluoropentyl)-1H-indazole-3-carbonyl]-valinate in illicit herbal products.

Synthetic cannabinoids, recently used as alternatives to Cannabis sativa, are among the most frequently abused drugs. Identified in 2014, the synthetic cannabinoids N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamide (5F-AB-PINACA) and methyl [1-(5-fluoropentyl)-1H-indazole-3-carbonyl]-valinate (5F-AMB) are carboxamides composed of 1-(5-fluoropentyl)-1H-indazole-3-carboxylic acid and valine amide/methyl ester. Because of their composition, these molecules have pairs of enantiomers derived from the chiral center of their amino acid structures. Previous studies on the identification of 5F-AB-PINACA and 5F-AMB did not consider the existence of enantiomers, and there have been no reports on the enantiopurities of synthetic cannabinoids. We synthesized both enantiomers of these compounds and then separated the enantiomers by liquid chromatography-high-resolution mass spectrometry using a column with a chiral stationary phase consisted with amylose tris (3-chloro-4-methylphenylcarbamate). Under the optimized conditions, the enantiomer resolutions were 2.2 and 2.3 for 5F-AB-PINACA and 5F-AMB, respectively. Analysis of 10 herbal samples containing 5F-AB-PINACA and one herbal sample containing 5F-AMB showed that they all contained the (S)-enantiomer, but the (R)-enantiomer was only detected in two samples and at a ratio of less than 20%.

Metabolism of the designer drug α-pyrrolidinobutiophenone (α-PBP) in humans: identification and quantification of the phase I metabolites in urine.

Urinary phase I metabolites of α-pyrrolidinobutiophenone (α-PBP) in humans were investigated by analyzing urine specimens obtained from drug abusers. Unequivocal identification and accurate quantification of major metabolites were realized using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry with newly synthesized authentic standards. Two major phase I metabolic pathways were revealed: (1) reduction of the ketone group to 1-phenyl-2-(pyrrolidin-1-yl)butan-1-ol (OH-α-PBP, diastereomers) partly followed by conjugation to its glucuronide and (2) oxidation at the 2″-position of the pyrrolidine ring to α-(2″-oxo-pyrrolidino)butiophenone (2″-oxo-α-PBP) via the putative intermediate α-(2″-hydroxypyrrolidino)butiophenone (2″-OH-α-PBP). Of the phase I metabolites retaining the structural characteristics of the parent drug, OH-α-PBP was the most abundant in all specimens examined. Comparison of the phase I metabolism of α-PBP and α-pyrrolidinovalerophenone (α-PVP) suggested a relationship between the aliphatic side chain length and the metabolic pathways in α-pyrrolidinophenones: the shorter aliphatic side chain (1) led to more extensive metabolism via reduction of the ketone group than via the oxidation at the 2″-position of the pyrrolidine ring and (2) influenced the isomeric ratio of a pair of diastereomers.

Simultaneous enantiomeric determination of MDMA and its phase I and phase II metabolites in urine by liquid chromatography-tandem mass spectrometry with chiral derivatization.

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) procedure was developed for the simultaneous determination of enantiomers of the prevalent designer drug 3,4-methylenedioxymethamphetamine (MDMA) and its phase I and phase II metabolites in urine with chiral derivatization. The analytes in urine were directly derivatized with chiral Marfey's reagent, N(α)-(5-fluoro-2,4-dinitrophenyl)-D-leucinamide, without extraction. The diastereomers of the N(α)-(2,4-dinitrophenyl)-D-leucinamide derivatives generated were determined by LC-MS/MS. Satisfactory chromatographic separation was achieved for the enantiomers of MDMA and its metabolites 3,4-methylenedioxyamphetamine, 4-hydroxy-3-methoxymethamphetamine (HMMA), HMMA glucuronide, and HMMA sulfate on a semimicro octadecylsilane column using linear gradient elution. With use of multiple reaction monitoring mode, the limits of detection of these analytes ranged from 0.01 to 0.03 µg/mL. Linear calibration curves were obtained for all enantiomers from 0.1 to 20 µg/mL in urine. The method showed sufficient reproducibility and quantitative ability. This is the first report of a simple LC-MS/MS-based analytical procedure with direct chiral derivatization in aqueous media that allows simultaneous enantiomeric determination of drugs and their metabolites, including glucuronide and sulfate derivatives.

Cross-reactivities of various phenethylamine-type designer drugs to immunoassays for amphetamines, with special attention to the evaluation of the one-step urine drug test Instant-View™, and the Emit® assays for use in drug enforcement.

Cross-reactivities of 76 kinds of phenethylamine-type designer drugs and related compounds to the urine drug tests Instant-View ™ (IV) (the Methamphetamine (MA) test, the Amphetamine 300 test, and the MDMA test) have been investigated. An on-site urine test kit consisting of these three IV tests has been evaluated for the on-site screening of MA users, and the kit has been found to have satisfactory specificity for drug enforcement purposes by separately detecting both MA and its metabolite amphetamine. The cross-reactivity profiles of Emit(®) II Plus Amphetamines Assay, Emit(®) II Plus Ecstasy assay, and Emit(®) d.a.u.(®) Amphetamine Class assay have also been investigated and discussed.

Metabolism and toxicologic analysis of tryptamine-derived drugs of abuse.

5-Methoxy-N,N-dialkyltryptamines are tryptamine derivatives that possess strong hallucinogenic effects. Because of their escalating popularity and potent physiological effects, an increasing number of acute poisoning cases have been reported in various countries. For their metabolism in humans, only a few studies have been reported. Thus, based on previous studies, the authors forecasted and synthesized authentic standards of their expected metabolites, which retained the structural characteristics of the parent drugs. Using these authentic standards, several urine specimens from abusers and rats were analyzed by gas chromatography mass spectrometry, high-performance liquid chromatography mass spectrometry, and high-performance liquid chromatography tandem mass spectrometry. The present study reveals that four metabolic pathways of great quantitative significance for 5-Methoxy-N,N-dialkyltryptamines to four characteristic metabolites, which retain structural characteristics identifiable with the parent compound, exist in humans and rats. The finding in the present study will be of great importance in the study on the metabolism of other psychotomimetic tryptamine-derived drugs of abuse and in forensic toxicologic analyses.

Determination of the metabolites of the new designer drugs bk-MBDB and bk-MDEA in human urine.

This is the first report on identifying the specific metabolites of the new designer drugs 2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one (bk-MBDB) and 2-ethylamino-1-(3,4-methylenedioxyphenyl)propan-1-one (bk-MDEA) in human urine using synthesized standards. Based on GC/MS and LC/MS, we identified N-dealkylation, demethylenation followed by O-methylation, and beta-ketone reduction as their major metabolic pathways. The quantitative analyses by LC/MS revealed that both demethylenation followed by O-methylation and beta-ketone reduction were superior to N-dealkylation and that both bk-MBDB and bk-MDEA were mainly metabolized into their corresponding 4-hydroxy-3-methoxy metabolites (4-OH-3MeO metabolites). After hydrolysis, the concentrations of 4-OH-3MeO metabolites and 3-hydroxy-4-methoxy metabolites of both bk-MBDB and bk-MDEA dramatically increased, suggesting that the metabolites mainly exist as their conjugates.

Determination of a newly encountered designer drug "p-methoxyethylamphetamine" and its metabolites in human urine and blood.

A newly synthesized designer drug, para-methoxyethylamphetamine (PMEA) was unexpectedly detected in the postmortem specimens of fatality involving drug intoxication in 2005, Japan. For unequivocal identification, the isomeric discrimination of PMEA and its positional-isomers was performed by GC/MS with the trifluoroacetylation. In order to prove the intake of PMEA, the characteristic metabolites of PMEA were also identified by GC/MS analysis of the urine specimen with trifluoroacetylation. As a result, para-methoxyamphetamine, para-hydroxyethylamphetamine (POHEA) and para-hydroxyamphetamine were identified as the major metabolites of PMEA. For the quantitative analyses of PMEA and its three metabolites in body fluids, an automated column-switching LC/MS procedure was developed, and applied to the postmortem blood and urine specimens. In this fatal case, blood concentration of PMEA was estimated to be 12.2 microg/mL and this level seemed extremely high in comparison with lethal blood-levels of its analogues, representing acute-intoxication of the victim. Based on the quantitative results, PMEA was found to be extensively metabolized to POHEA via O-demethylation, partly followed by its conjugation.

Discrimination and identification of the six aromatic positional isomers of trimethoxyamphetamine (TMA) by gas chromatography-mass spectrometry (GC-MS).

A reliable and accurate GC-MS method was developed that allows both mass spectrometric and chromatographic discrimination of the six aromatic positional isomers of trimethoxyamphetamine (TMA). Regardless of the trifluoroacetyl (TFA) derivatization, chromatographic separation of all the investigated isomers was achieved by using DB-5 ms capillary columns (30 m x 0.32 mm i.d.), with run times less than 15 min. However, the mass spectra of the nonderivatized TMAs, except 2,4,6-trimethoxyamphetmine (TMA-6), showed insufficient difference for unambiguous discrimination. On the other hand, the mass spectra of the TFA derivatives of the six isomers exhibited fragments with significant intensity differences, which allowed the unequivocal identification of all the aromatic positional isomers investigated in the present study. This GC-MS technique in combination with TFA derivatization, therefore, is a powerful method to discriminate these isomers, especially useful to distinguish the currently controlled 3,4,5-trimethoxyamphetmine (TMA-1) and 2,4,5-trimethoxyamphetmine (TMA-2) from other uncontrolled TMAs.

Direct determination of glucuronide and sulfate of 4-hydroxy-3-methoxymethamphetamine, the main metabolite of MDMA, in human urine.

A sensitive and reliable LC-ESI-MS procedure for the simultaneous determination of MDMA and its five metabolites including 4-hydroxy-3-methoxymethamphetamine (HMMA) conjugates has been established following the synthesis of two HMMA conjugates, 4-hydroxy-3-methoxymethamphetamine-glucuronide (HMMA-Glu) and 4-hydroxy-3-methoxymethamphetamine-sulfate (HMMA-Sul). Pretreatment of urine samples with methanol and LC-MS employing a C(18) semi-micro column with a gradient elution program provided the successful separations and MS determinations of these analytes within 20 min. Upon applying the method to MDMA users' urine specimens, HMMA-Glu and HMMA-Sul have been directly determined, suggesting the superiority of sulfation to glucuronidation in the HMMA phase II metabolism.

Metabolism of the psychotomimetic tryptamine derivative 5-methoxy-N,N-diisopropyltryptamine in humans: identification and quantification of its urinary metabolites.

The urinary metabolites of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) in humans have been investigated by analyzing urine specimens from its users. For the unequivocal identification and accurate quantification of its major metabolites, careful analyses were conducted by gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and liquid chromatography-tandem mass spectrometry, using authentic standards of each metabolite synthesized. Three major metabolic pathways were revealed as follows: 1) side chain degradation by O-demethylation to form 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT), which would be partly conjugated to its sulfate and glucuronide; 2) direct hydroxylation on position 6 of the aromatic ring of 5-MeO-DIPT, and/or methylation of the hydroxyl group on position 5 after hydroxylation on position 6 of the aromatic ring of 5-OH-DIPT, to produce 6-hydroxy-5-methoxy-N,N-diisopropyltryptamine (6-OH-5-MeO-DIPT), followed by conjugation to its sulfate and glucuronide; and 3) side chain degradation by N-deisopropylation, to the corresponding secondary amine 5-methoxy-N-isopropyltryptamine (5-MeO-NIPT). Of these metabolites, which retain structural characteristics of the parent drug, 5-OH-DIPT and 6-OH-5-MeO-DIPT were found to be more abundant than 5-MeO-NIPT. Although the parent drug 5-MeO-DIPT was detectable even 35 h after dosing, no trace of its N-oxide was detected in any of the specimens examined.